The post-translational modifications of proteins are known to enhance heterogeneity among proteins. For instance several minor hemoglobins, designated AIa1, AIa2, AIb, AIc, AId and AIe, are present in red cell hemolysates from human adults. Hb AIc has a hexose moiety and Hb AIa1 and Hb AIa2 has glucose-6-phosphate (G6P) or fructose 1,6-diphosphate, attached to the amino-terminus of beta chains. The minor hemoglobins present in the red cells of newborns (Hb FI) contain mainly N-acetylated gamma chains. However, the presence of glycosylated Hb F cannot be ruled out and preliminary data indicates that this indeed is true. It is not known whether Hb FI in adults is primarily acetylated as in newborns or glycosylated as Hb AIc in adults. The acetylation of hemoglobin seems to be enzyme dependent in contrast to nonenzymatic glycosylation. Acetyltransferase activity has been detected in the reticulocyte lysates of chicken and newborn. During the proposed study the extent of heterogeneity of minor hemoglobins present in the red cells of newborns and adults with elevated Hb F will be determined. Various chromatographic procedures will be applied to separate, isolate and purify minor hemoglobins. They will be characterized by determining N-acetyl groups, protein bound organic phosphates, and sugar moieties and by functional studies. The kinetics of synthesis of the Hb FI's will be studied by incubating reticulocytes from the newborns and from the adults with 14C-leucine with and without protein synthesis inhibitors and by pulse-chase studies. Hb A, Hb S and Hb F will be glycosylated in vitro with 14C-labelled glucose and G6P. Acetyltransferase will be prepared and purified from the reticulocytes of chicken, cat and umbilical cord blood. The regulatory factors and the specificity of this enzyme in acetylating various hemoglobins will be determined using cell-free acetylating systems. An assay system for this enzyme will be developed and reticulocytes and red cells from various sources will be tested. Both in vivo and in vitro glycosylated and acetylated hemoglobins will be used to identify the derivatized amino acid residues.